1. Field of the Invention
The present invention relates to a method for monitoring the generation of oxidative products from cell-biomaterial interactions. More specifically, the present invention relates to a chemiluminescence method for continuously monitoring in real time the generation of oxidative products from cell-biomaterial interactions in vitro.
2. Description of Related Art
With the improvements in health and increased life span in the United States population, comes the need for improved reliability of implanted medical devices, particularly those which are used for life-supporting structural and organ systems. Up to now, the expected life time for these devices was on the order of 10 years. As in the case of the prosthetic heart valves, pacemakers, orthopedic prostheses, etc., patient survival is extended for longer periods necessitating a corresponding improvement in reliable performance requirements (Kambic et al., "Biomaterials in artificial organs," Chem. and Eng. News, Apr. 14, pp. 31-49, 1986). In fact, the types of problems associated with long term usage are quite different from those of short term usage.
Performance parameters fall into two general categories: materials properties and dynamic function. Materials properties are tested by the classical methods of physico-chemical characterization and dynamic function is tested in simulated environments with accelerated life tests. The compatibility of the devices and their materials with the host, the human body, is tested with a classical battery of biocompatibility tests associated with the intended site of use (ASTM Standard 748-82, British Standards Institute No. 5736, and Canadian Standards Association CAN3-Z310.6-M84, September 1984; Williams, Definitions in Biomaterials, Proceedings of a consensus conference of the European Society for Biomaterials, Chester, UK, Mar. 3-5, 1986, Elsevier Press, 1988). These tests include acute and chronic toxicity determinations in experimental animals and mutagenic and carcinogenic tests in animals and tissue culture. Long term testing of any kind is expensive and often requires use of a variety of different animal models. Manufacturers will have their products or components tested by a battery of tests chosen according to selected criteria (North American Science Associates, Incorporated (NAmSA) Safety Evaluation Guidelines, 1989).
The data required to document extended usage in the human are either extrapolated from controlled studies of limited duration or result from analysis of a limited number of explanted devices. These are usually explanted due to infection or trauma at the site, or failure of the device or an unrelated death of the patient (Chawla et al., "Degradation in polyurethane pacemakers leads," 12th Annual Meeting of the Society for Biomaterials, Minneapolis-St. Paul, Minn., 1986). Unfortunately, data collected in this manner do not constitute well-controlled studies.
Approaches to validating the in vivo performance life time of materials and devices include analyses of the interface between the host and the material of the device. Much of this is aimed at determining the biocompatibility of the material with blood or blood components and also with assessing the toxicity of leachables from the material. Increasingly, discussion is starting to focus on measurement of material degradation after implantation. Biological model systems and methods of monitoring and predicting the degradation are needed for each type of material and site of use of the devices, as the host response will vary with these two parameters.
Some of the properties of materials that are reflective of their performance potential have been studied: surface characterization as hydrophobic or hydrophilic by measurement of the contact angle, crystallinity of the material, lipophilic nature, (Fulghum et al., "Surface characterization," Anal. Chem., 61, pp. 243R-269R, 1989; Ratner et al., "Biomaterial surfaces," J. of Biomedical Materials Research: Applied Biomaterials, 21 (A1), pp.59-90, 1987; National Heart, Lung and Blood Institute Working Group Guidelines for Blood-Biomaterial Interactions, 1985), mineralization (Stokes et al., "Environmental stress cracking in implanted polyurethanes," 2nd World Congress on Biomaterials, Wash., DC, 1984) and thrombogenicity (Schoen, F. J.: Interventional and Surgical Cardiovascular Pathology: Clinical Correlations and Basic Principles, W. B. Saunders, Philadelphia, 1989, pp. 1-415), and phase transition (Wilkes and Emerson, J. Applied Physics, 47:4261, 1976).
At present there is very little routine testing performed to determine the effect of the host response on the device and it's materials. An understanding of the interaction between these materials and the body's environment is fundamental to the prediction of long term functional performance. This is due to the fact that the nature of the environment is difficult to determine and duplicate in vitro.
The research literature shows that all materials elicit a reaction from the human body. This reaction is purposeful: to attack, destroy and remove the invading device. There are several chemical mechanisms of degradation for a material in the bioenvironment. These include: mineralization, specifically calcification (Hennig et al., "Calcification of artificial heart valves and artificial hearts," Proc. Eur. Soc. Artificial Organs, 8, pp. 76-80, 1981), hydrolysis including enzymatic hydrolysis (Ratner et al., "In vitro studies of the enzymatic biodegradation of polyetherurethanes," Abstract: 12th Annual Meeting of the Society for Biomaterials, Minneapolis-St. Paul, Minn., 1986; Chu et al., "The effect of gamma irradiation on the enzymatic degradation of polyglycolic acid absorbable sutures," J. Biomedical Materials Res., 17, pp. 1029-1040, 1983; Smith et al., "The enzymatic degradation of polymers in vitro," J. Biomedical Materials Res., 21, pp. 991-1003, 1987; Williams, "Some observations on the role of cellular enzymes in the in vivo degradation of polymers," Corrosion and Degradation of Implant Materials. ASTM, ASTM Special Technical Publication 684, B. C. Syrett and Acharya, Eds. Philadelphia, pp. 61-75, 1978), and/or oxidation. These processes when coupled with a static or dynamic mechanical stress induced, for example, by implant fixation technique or motion, result in structural changes in the material as in the case of polyurethane insulated pacemaker leads (Stokes et al., 1984). This would manifest itself as a cracked or crazed surface and results in the loss of insulating properties of the polyurethane. Knowledge of the role of enzymes, of other biochemical compounds and of cells in inducing such mechanisms is important in devising means for protecting materials during long term implantation. Since many polymeric implant materials have molecular sites which can be oxidized, this may be a principle means of degradation.
The initial response to an invasive device is described as the inflammatory reaction with subsequent wound healing (Marchant et al., "Biocompatibility and an enhanced acute inflammatory phase model," Corrosion and Degradation of Implant Materials: Second Symposium. ASTM STP 859, A. C. Fraker and C. D. Griffin, Eds., ASTM, Philadelphia, pp. 251-266, 1985; Marchant et al., "in vivo biocompatibility studies, VII, Inflammatory response to polyethylene and to a cytotoxic polyvinylchloride," Journal of Biomedical Materials Research: 20, pp. 37-50, 1986). One of the first systems activated in the inflammatory reaction is the complement cascade. Recent evidence indicates that the clotting mechanism or complement cascade can be modulated by the type of material employed (Kiyosawa et al., "Effects of Intraocular Lens Materials on Complement Activation and Macrophage Function, Nippon Ganka Gakkai Zasshi; 92(4), pp. 603-610, 1988) These results are interpreted to mean that there are specific sites or chemical groups on certain materials that cause activation of parts of the complement pathway. Certain materials such as silk sutures and nylon fibers suppress the complement cascade (Zimmerli et al., "Pathogenesis of Foreign Body Infection: Description and Characteristics of an Animal Model," Journal of Infectious Diseases, 146(4), pp. 487-497, 1982; Zimmerli et al., "Comparative Superoxide-Generating System of Granulocytes from Blood and Peritoneal Exudates," Infection and Immunity. 46(3), pp. 625-630, 1984; Zimmerli et al., "Pathogenesis of Foreign Body Infection: Evidence for a Local Granulocyte Defect," Journal of Clinical Investigation, 73, pp. 1191-1200, 1984). There is the possibility that complement results in an oscillatory reaction which continually attracts the leucocytes to the implant site. Analyses which demonstrate this phenomenon could be very useful in the prediction of material performance in the host.
The primary cells attracted during the initial phase of the inflammatory response are the polymorphonuclear leucocytes with their secretions of lysozymes and hydrolytic and oxidative enzymes and oxidative oxygen products such as hydrogen peroxide, superoxide anion hydroxyl radical, and hypochlorous acid (Allen, "Phagocytic leukocyte Oxygenation Activities and Chemiluminescence: A Kinetic Approach to Analysis," In: Methods in Enzymology: Bioluminescence and Chemiluminescence, Part B, Volume 133, Marlene A. DeLuca and William D. McElroy, Eds., Academic Press, Inc., New York., pp. 449-493, 1986; Thompson et al., "Oxygen metabolism of the HL-60 cell line: Comparison of the effects of monocytoid and neutrophilic differentiation," J. Leukocyte Biology. 43, pp. 140-147, 1988; Cohen et al., "Phagocytes, O-2 reduction and hydroxyl radical," Reviews of Infectious Diseases, 10(6), pp. 1088-1096, 1988). This arsenal of chemicals can effect the material and eventually degrade it to the point of malfunction. Subsequent to this acute response, macrophages are differentiated and attracted to the site, via cytokines and lymphokines, where they too attempt to destroy the foreign material. Adaptive processes have evolved to enable both cell types to engulf by phagocytosis particulate invaders such as microorganisms and to destroy them by secretion of energetic, metabolic oxidative products and enzymes within the phagosome. Concomitant with this response is the utilization of increased amounts of metabolic oxygen, referred to as the respiratory burst (LKB Wallac, "The LKB Wallac 1251 luminometer--A Tool for the detection of opsonophagocytic dysfunctions," Product News: Luminescence Analysis. pp. 1-6, 1985). Although it is thought that the oxidative oxygen products are primarily responsible for killing of intracellular parasites, it is known that these macrophages can still kill organisms such as trophozoites without the utilization of additional oxygen-using mechanisms. Such non-oxidative microbiocidal functions are currently under investigation by a number of researchers, i.e., the study of defensins (Elsbach and Weiss, "Chapter 24: Phagocytic Cells: Oxygen-independent antimicrobial systems." In: Inflammation: Basic Principles and Clinical Correlates. John I. Gallin, Ira M. Goldstein and Ralph Snyderman, 1988).
When the material is too large for engulfment, the macrophage enlarges into a multinucleated cell, termed a foreign body giant cell. This process is termed "frustrated phagocytosis" (Ziats et al., "In vitro and in vivo Interactions of Cells with Biomaterials," Biomaterials. 9, pp. 5-13, 1988), since the macrophages cannot actually engulf the very large invader, in this case the material of the implant. These cells attach to the material and may cause damage due to extracellular secretions. This chronic phase may continue for extended periods. The subsequent formation of a fibrous capsule around the device results in a "walling-off" or exclusion of the device from the biological milieu. In order to study the biological response without this exclusion and be able to sample by aspiration, cellular populations at the site, researchers in this area have used a stainless steel cage to hold the implant materials to be tested (Marchant et al., "In vivo biocompatibility studies, I, The cage implant system and a biodegradable hydrogel," Journal of Biomedical Materials Research, 17, pp. 301-325, 1983; Marchant et al., "Preliminary cell adhesion and surface characterization studies," Journal of Biomedical Materials Research. 18, pp. 309-315, 1984; Marchant et al., "In vivo biocompatibility studies, V, In vivo leukocyte interactions with Biomer," Journal of Biomedical Materials Research. 18, pp. 1169-1190, 1984; Marchant et al., 1986). These studies produced much information on cellular aspects of the inflammatory reaction. Studies with biomedical materials have shown changes in the cellular supernatant secretions that contain fibroblastic and thymocyte proliferative Interleukin-1-like activity (Miller et al., "Characterization of biomedical polymer-adherent macrophages: Interleukin 1 generation and scanning electron microscopy studies," Biomaterials, 10, pp. 187-196, 1989; Miller et al., "Human monocyte/macrophage activation and interleukin 1 generation by biomedical polymers," Journal of Biomed. Mater. Res. 22, pp. 713-731, 1988; Miller et al., "Generation of IL1-like activity in response to biomedical polymer implants: A comparison of in vitro and in vivo models," J. Biomed. Res., 23, pp. 1007-1026, 1989). Zimmerli et al. (1982, 1984) have used cages composed of teflon and of PMMA to minimize the inflammatory response to the test holder of the material.
Conventional biocompatability tests measure cell death, cell damage, mutagenicity or other cytopathology at the site of implantation into test animals or tissue culture vessels. These measures do not distinguish among the various cell responses and are subjectively quantified, at best. Knowledge of the amount and rate of production of oxidative products in response to different materials is desirable since it could serve as an indicator of a material's stability in the biological environment and could be useful in predicting the long term reliability of the material. Biological oxidizing agents include: H.sub.2 O.sub.2, O.sub.2.sup.-, OH or HOCL (Castranova et al., Chapter 1: "Chemiluminescence from Macrophages and Monocytes. Cellular chemiluminescence," In: Cellular Chemiluminescence, Volume II, Knox Van Dyke and Vincent Castranova, Eds. CRC Press, Boca Raton, Fla., pp. 3-19, 1987; Allen, 1986; Lilius et al., "Chemiluminescence emission from enriched fraction of human natural killer cells," Analytical Applications of Bioluminescence and Chemiluminescence. L. J. Kricka, P. E. Stanley, G. H. G. Thorpe and T. P. Whitehead, Eds., Academic Press, New York, pp. 397-400, 1984; Lilus et al., "A very sensitive and rapid chemiluminescence method for the measurement of phagocytosis," Analytical Applications of Bioluminescence and Chemiluminescence, L. J. Kricka, P. E. Stanley, G. H. G. Thorpe and T. P. Whitehead, Eds., Academic Press, Inc., New York, pp. 401-404, 1984). It is therefore desired to be able to continuously monitor the production of some of these oxidants with a real time non-destructive method. Conventional technology may attempt to use chemical or spectrophotometric assays to determine the amount of oxidative products instantaneously produced in response to introduction of a material in the biological environment. However, the short lifetime of the oxidative product and the fact that they are continuously produced by the cells introduces errors into these methods. In some cases, the presence of the opaque biomaterial interferes with the assay and removing the test cells from the material is difficult and not quantitative. The cells are thus perturbed and the subsequent measures fail to be true to the original biological conditions.